Biophysical Perspective
Optics-Integrated Microfluidic Platforms for Biomolecular Analyses Kathleen E. Bates1,2 and Hang Lu1,2,* 1 Interdisciplinary Program in Bioengineering and 2School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia
ABSTRACT Compared with conventional optical methods, optics implemented on microfluidic chips provide small, and often much cheaper ways to interrogate biological systems from the level of single molecules up to small model organisms. The optical probing of single molecules has been used to investigate the mechanical properties of individual biological molecules; however, multiplexing of these measurements through microfluidics and nanofluidics confers many analytical advantages. Optics-integrated microfluidic systems can significantly simplify sample processing and allow a more user-friendly experience; alignments of on-chip optical components are predetermined during fabrication and many purely optical techniques are passively controlled. Furthermore, sample loss from complicated preparation and fluid transfer steps can be virtually eliminated, a particularly important attribute for biological molecules at very low concentrations. Excellent fluid handling and high surface area/volume ratios also contribute to faster detection times for low abundance molecules in small sample volumes. Although integration of optical systems with classical microfluidic analysis techniques has been limited, microfluidics offers a ready platform for interrogation of biophysical properties. By exploiting the ease with which fluids and particles can be precisely and dynamically controlled in microfluidic devices, optical sensors capable of unique imaging modes, single molecule manipulation, and detection of minute changes in concentration of an analyte are possible.
INTRODUCTION Despite the burgeoning activity in the field of optofluidics, there are many open opportunities to integrate optical tools with biological microfluidic handling systems to perform previously impossible analyses. Many optics-integrated microfluidic systems demonstrate comparable sensitivity to nonmicrofluidic systems, but most do not take full advantage of the potential to integrate sample handling and parallelize assays for ease of biological analysis. Assay systems that simplify and multiplex measurements and diagnostics could expand the reach and impact of optofluidic systems by making some types of high-throughput measurements possible and by making technology available to more diverse settings. Biological samples are diverse yet sparse solutions of cells, proteins, and small molecules, and are typically acquired in limited fluid volumes (milliliters to microliters), making them especially difficult to process. The issues of sample complexity and low molecular concentration, however, can be significantly decoupled from the problem of detection and quantification of analytes by incorporating microfluidic
methods. Myriad developments in microfluidics over the past two decades have demonstrated that biological samples can be successfully manipulated and separated from undesired components on-chip. The inherent heterogeneity in biological systems is a complicating factor that often requires the same assay be performed many times, a job that microfluidics are well suited for. In addition, many optical sensing systems can detect low concentrations of analyte, often with limits of detection in the pico- to attomolar range. In this review, we will attempt to provide the reader with an introduction to biological sample preparation in microfluidics as well as available cellular and subcellular techniques for optical manipulation and sensing in the hope to inspire new and interesting marriages between these fields. To this end, we will discuss manipulations and separations at molecular and cellular levels using optical techniques, hydrodynamics, and other force fields, in addition to optical tools commonly used in sensing systems, and optical sensing systems themselves. MATERIALS AND METHODS
Submitted October 13, 2015, and accepted for publication March 8, 2016. *Correspondence: [email protected] Editor: Brian Salzberg. http://dx.doi.org/10.1016/j.bpj.2016.03.018 Ó 2016 Biophysical Society
1684 Biophysical Journal 110, 1684–1697, April 26, 2016
Physical separations Separations are often required when working with biological samples to isolate a desired entity, sort a mixed population, or concentrate highly dilute
Optofluidics for Biomolecular Analyses mixtures. Because of the large variety of biological molecules that exist in cellular environments and the rarity of many of those molecules, separation is frequently the first step in biological analysis. By integrating these separation steps into a microfluidic system, it is possible to perform efficient and highly precise separations over a large range of particle sizes, from individual molecules and proteins (on the order of angstroms) to cells (on the order of micrometers) to small model organisms such as Caenorhabditis elegans (on the order of millimeters), in small volumes of liquid (on the order of nanoliters). Optical separations by themselves are limited by the analytical meaningfulness of optical differences. For example, the refractive index of individual cells within isogenic cell populations is often highly heterogeneous, depending on size and shape, making separation difficult. Consequently, although high-throughput and single-cell optical separation has proven very useful, there remain opportunities to improve the large-scale handling of particles. Before delving into more specialized optical separations, we will briefly mention some relevant nonoptical separations that supplement optical separations and could well be used to complement other optical sensing techniques. For a more thorough review of individual techniques, we point the reader to references cited in this section.
Nonoptical separations Within nonoptical separations, there are primarily two types of separation: those that occur passively due to hydrodynamic forces (pressure fields) within a microfluidic device, and those that occur due to imposed electrical or magnetic fields. As hydrodynamic forces are intrinsically required for microfluidic flow, they are particularly easy to integrate as a means of separation. The laminar nature of microfluidic flow results in a replicable system whose flow field can be modeled relatively simply, dramatically streamlining device design. Cells or other particles with different size, shape, or stiffness passing through a specifically designed flow field experience differential forces that direct them to different regions of the flow, allowing them to be separated passively. Applications are typically limited to populations where physical properties contrast significantly, but recent developments in inertial microfluidics have shown that micro- and nanoparticles can be separated in a high-throughput manner (600 ml/min) with high purity (90–99%), demonstrating potential for cell throughput close to the order of magnitude of flow cytometers (1,2). Although inertial microfluidics operate at higher Reynolds numbers, they remain laminar. Despite exposure of biologicals to higher shear stress compared with noninertial microfluidics, shear forces are much lower than what samples experience when they are pipetted, and separation in this manner may be gentler than centrifugation purification or physical filtration. Operation windows of inertial devices, however, are quite small and careful design is required (3). Microfluidic chromatography is another common method for which implementation is straightforward. High-resolution size separation over a large size range can be performed using deterministic lateral displacement, where regularly spaced features within the microfluidic channel bump different sized particles into different streamlines (4,5). For biological entities with a defined immunophenotype, microfluidic channel surfaces may be coated with antibodies before sample addition to perform affinity chromatography (6). The concept is simple, however, the requirement for a sample with a defined immunophenotype may be limiting, as cells must come into sufficient contact with channel walls to be captured, and antibodies are not only costly, but may also exhibit nonspecific binding. Externally applied fields may also be applied to microfluidic channels using principles of field flow fractionation (7). Separation by magnetic field is possible, but requires the use of magnetically responsive particles and antibodies in addition to an external magnetic field (6). Often the most useful technique in this category is dielectrophoresis (DEP) (8). Differences in dielectric properties of sample constituents allow continuous separation using an inhomogeneous electric field in DEP. The primary challenge of DEP is that many characteristics can impact the dielectric properties of the cell, including, but not limited to, cell size, and cell membrane and cytosolic content (which impacts permeability, capacitance, and conductivity). How-
ever, DEP has the significant advantage of being label-free and a reasonably sensitive method for separation despite its nonspecific nature (9–14). The applicability of DEP must be weighed carefully, as more complicated design considerations are involved, and exposure to an electrical field may damage or alter biological samples.
Optical property-based separations Label-free methods In general, optical manipulations operate on objects of approximately micron scale and have high spatial resolutions in addition to being labelfree and operating in a noncontact mode (15,16). Techniques that require labels have certain disadvantages; interference with molecular structures whose physical conformation is desired and interaction with biological processes of interest are both potential sources of error in labeled techniques, not to mention the added expense of additional reagents. The use of optical manipulation techniques on-chip often still requires bulky external equipment and patient optical alignment, however, external optics also allow optics to be decoupled from the microfluidics, enabling on-the-spot adjustment of key parameters rather than requiring further fabrication and redesign. Similar to DEP, optical manipulation can be very precise, but may require more complicated fabrication or alter cell physiology when highpowered lasers are involved. Investigating physical properties on the single-cell and single-protein level is an ideal application point for integrating optics and microfluidics. The use of optical tweezers (OT) to apply force, torque, and other fine-scale manipulations in a massively parallel fashion provides a window into the world of heterogeneity in both mechanical and optical senses. OTs use spatial light gradients to focus micron-sized particles to the center of a laser beam due to differences in radiation pressure. Classic examples are the measurement of the movement of the biological motor kinesin, and the characterization of the elasticity of DNA through attachment of these nanoscale objects to micrometer-sized spheres (17–19). Typical OT set-ups require high-power infrared lasers to focus particles, necessitating substantial modification to commercial microscopes and limiting the number of manipulable entities. The high-powered lasers needed could also lead to photodamage of biological molecules. Recently developed microfluidic alternatives for OTs have used plasmonic nanostructures to create evanescent waves at the interface of thin metal surfaces, generating a very strong near-field effect capable of trapping dielectric particles (20,21). Due to the relative system-level optical simplicity of optics on microfluidic systems in comparison to OTs, the localized nature of near-field effects, and the overall compactness of systems relying on surface plasmon resonance (SPR), plasmonics-based trapping lends itself to integration with microfluidic devices. SPR techniques on-chip is very promising for precise particle manipulations, including subdiffraction-limit trapping (22). Efficient assay parallelization leads to greater ease of sampling larger subsets of a population, thereby informing our perspective on heterogeneity. A current limitation for optical manipulations is the inefficiency of scaling up to many optical traps on a single microfluidic device. Although technologies like holographic optical tweezers and opto-electronic tweezers allow large-scale manipulation, these systems are expensive and optical contrast between different types of cells is often low, making manipulation difficult and again leading to the use of high power lasers (23,24). In a similar vein, projection of optical images onto microfluidic devices have been used to passively separate cells based on their optical contrast and polarizability (which increases as size increases), however, this relies on changes in optical contrast and polarizability being useful and meaningful ways to separate cells, which is not always the case (25). A technique that may enable high-throughput and precise manipulation on-chip has recently been described that employs lower power lasers than those typically required for multiplexing optical traps (26). In this case, multiple traps are created at antinodes of a standing wave from a single laser
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Bates and Lu and traps are repositioned by using thermal energy to change the phase of light waves. Rudimentary sorting has been performed by holding bead-conjugated biological samples in one set of traps while applying small lateral pressure fields to push untrapped particles out of the device, or alternatively, by flow switching based on Raman spectra obtained from single cells (16,26). This application relies heavily on the creation of highly stable traps that would experience significant drift outside of a well-defined microfluidic environment, and the ability to switch fluid flow quickly, a clear strength of microfluidic systems. Continued effort to create large arrays for simultaneous manipulation of individual particles in an efficient manner will likely remain a significant goal because of its potential impact on easing the investigation of heterogeneity and other fields reliant on big data for answers, for example genomic studies.
Labeled separations Images are one of the highest content data formats; a typical photosensor has several megapixels worth of data points. The conceptually simple idea of separation based on image properties and features is an integral part of many biological assays that can be supplemented through the use of microfluidic devices. By using microfluidic chips, micrometer-scale specimens can be imaged either in series or in parallel, both of which can enable high-throughput modalities (27). This is a common theme over a broad range of phenotyping applications, including both small organisms such as C. elegans as well as cells (28–30). The ability to restrict the movement of cells and model organisms while orienting asymmetrical objects reproducibly via hydrodynamic forces within microfluidic devices eases the time burden of physical manipulation as well as simplifies quantitative comparison of specimens. For living animals, various schemes are available for immobilization within devices, including cooling and compression, which are often much gentler compared with the usual glue or anesthetic methods (29,31). Because these chips are made of the flexible polymer polydimethylsiloxane (PDMS), chips are inexpensive to make, optically transparent, and compatible with biological specimens. The material properties of PDMS also enable on-chip membrane valves to conveniently control fluid flow. Although the use of microfluidics in this manner requires additional pressure sources and controls, time involved in operation and handling is orders of magnitude less compared with individually mounting and imaging animals or cells. Thus, for high-throughput experiments or experiments requiring large numbers of samples, as in genetic screens and phenotyping experiments, more comprehensive arrays of experiments come within reason for a single researcher to perform. Many separations based on image properties are facilitated by fluorescent labeling, but separation through image-based phenotyping is not exclusively confined to labeled reagents. Given that an object in a population to be sorted has sufficient regional variation that can be identified via brightfield, darkfield, or other modes of microscopy, these types of images can be used as the basis for separation. However, the use of transgenic cells and species that encode fusion proteins for reporter systems are common because of the ability to label proteins in subpopulations of cells in vivo. Subcellular localizations of proteins can often be determined as well (although how significantly fusion proteins affect localization is a matter of debate), and autofluorescence is typically low enough in most cells and organisms that differentiating background from foreground is relatively simple. Compared to a label-free system, a subcellularly localized reporter system can give much more detailed information about whole-system function because tagged proteins are actively involved in cellular processes. In addition, the number of reporters is theoretically only limited by the number of optically orthogonal reporters possible within the color spectrum, although in practice excitation and emission spectra often overlap significantly, making it impractical to use more than three or four reporters. This has recently driven the development of reporters that emit in nearinfrared range. Despite the usefulness of fluorescent reporters, the effort required to transform organisms and transfect cells can be significant, and damage due to photobleaching, as well as differences in the rate of photobleaching at different locations within the cell makes long-term moni-
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toring more challenging for parallelized methods (32). If intracellular or intraorganismal protein distribution is not required, this additional effort may not be warranted and label-free sensing may be more appropriate. On microfluidic chips, active separation of samples may involve acquisition of an image of the specimen, followed by actuating a valve on device based on optical feedback (i.e., based on specific image features), limiting the specimen to following a single path of least fluidic resistance. Recovery of specific specimens out of a large microfluidic array is sometimes only possible through the use of optical methods like OT that can provide control over individual specimens, however, this increases cost and power input significantly while requiring more user intervention (33). In contrast to most optical separation methods, using image features as criteria for separation is extremely versatile because separation need not be based upon a single feature, such as refractive index or physical size, but upon combinations of many features predefined by the user. Machine learning algorithms may be used to produce these combinations, limiting user-introduced bias toward phenotypes most evident to the human eye. However, even simple image processing followed by the application of heuristics relevant to the application of interest can be of use in situations where a desired phenotype is predefined (33). Although computer vision tools vary in terms of ease of use, there are many machine learning techniques that can be used out-ofthe-box or with little modification. The reader may consult Elicieri et al. (34) for more insight into image analysis packages and image processing and computer vision examples. In addition to consumer software available for computer vision, continuing innovations in computer vision algorithms have resulted in consistent improvement in terms of accuracy, speed, and bias reduction in the ability to detect image features in an unsupervised manner.
RESULTS Optical tools for guiding light on-chip In many optical sensing situations, the integration of multiple optical tools, such as lenses, waveguides, and lasers is critical to create a mechanism for sensing. Traditional optical systems for use with biomicrofluidics rely heavily on these components in the form of off-chip microscopy. In comparison, although most of the on-chip techniques described here are still dependent on external sensors (e.g., charge-coupled device or complementary metal oxide semiconductor sensors) to collect data, on-chip and partially on-chip optics confer advantages in terms of footprint, cost, and timescale while maintaining sensitivity expected from similar techniques. As many high-sensitivity optical techniques already require micro- or nanoscale structures to confine and propagate light, some type of microfabrication is already necessary in most cases. The addition of microfluidics, then, follows logically as a convenient way to control sample volume and handling without adding significantly more fabrication steps. On-chip lens systems Lenses are integral parts of most optical microscopy systems, but traditionally have little flexibility in terms of dynamic changes to focal length, necessitating precise translations in the z-direction to collect in-focus light from multiple image planes. In contrast to the rigidity of traditional lenses, cheaply produced on-chip lenses provide flexibility
Optofluidics for Biomolecular Analyses
both in terms of chip design and material properties, and can be used to facilitate on-chip imaging in a variety of ways. Practically, lenses are some of the easiest elements to integrate, as many common materials in microfabrication can be manipulated into cheap and robust lens arrays using simple techniques (35,36). Particularly in photometry applications, where wavefront fidelity is less important, simple converging and diverging lenses can be of use. Although variable-focus lenses were initially developed at the macroscale for use in spectacles, the fine scale at which they can be fabricated in microfluidics has yielded a small host of applications at the micron scale (37–39). Adjustable focal length microfluidic lenses are tunable by variation of refractive indices or lens shape. Refractive indices may be controlled through lens composition, or via thermal or electric fields, can be tuned pneumatically, through environmentally responsive materials such as hydrogels, or with electromagnetic fields (38–48). A primary challenge in fabricating variable focus lenses that function by lens deformation is in creating lenses with large dynamic ranges and short response times; for example, pneumatic control of fluid-filled reservoirs behind thin, flexible membranes (composed of, for example, PDMS) is a common microlens approach that is limited by slow response times despite large tuning ranges and easy device integration (Fig. 1 A) (49–51). Although PDMS is optically transparent and has low losses in the visible range, light scattering through the polymer is not always negligible due to nanoparticles of silica within commercially available products (52). Alternatively, nanoliter-size droplets can be easily, robustly, and precisely formed in microfluidic devices whose focal length can be tuned by altering the surface tension at the droplet interface or electrical field within the microfluidic device (43,49). Environmentally responsive lenses, such as hydrogels, have also been used to adjust focus in a passive
A
manner (see Fig. 1 D). In one interesting example, red blood cells were used as microlenses, switching their focal length from negative to positive values by changing the chemistry of the buffer solution and using their focusing properties as a basis for phenotyping (53). Using electrowetting to induce changes in lens geometry by altering the liquid contact angle has distinct advantages in consistency of fluid deformation, response time, and the need for only small driving voltages as compared with large pressures (Fig. 1 B) (45,46,54). The creation of arrays of these lenses on-chip is simplified by the ability to address individual droplets to spatial locations, either hydrodynamically or through the use of electrophoresis or electrowetting on dielectrics (EWOD). Temporal modulation of an electric field has recently been used to both create two-dimensional (2D) monodisperse droplet arrays for use as microlenses and to achieve large deformation of microlenses for variable-focus tunability over millisecond timescales, a speed similar to that of the inertial response limit that can be affected by a common piezoelectric z-stepper (49,55). In addition to these vertical tunable lenses, tunable lenses composed of core and cladding fluids within horizontal microfluidic channels also enable dynamic modulation of lens shape through hydrodynamics or electrokinetics (Fig. 1 C) (47,56–58). By exchanging fluids used for core and cladding, an additional parameter can be used for focal plane adjustment, further broadening the capabilities of this type of variable focus lens (59). Although these lenses are often easier to initially form, they can also be perturbed by small particles or bubbles within the flowing fluid, leading to significant chromatic and geometric aberrations and making them more difficult to maintain over long periods. Table 1 summarizes advantages and disadvantages of the primary types of dynamic lenses and example applications. Although acoustic tuning of microfluidic lenses has not been explored to our
cladding core
C
FIGURE 1 Physical configuration of variable focus lenses. (A) Pneumatically tuned lens. (B) Lens controlled by electrowetting. (C) Hydrodynamic lens. (D) Environmentally responsive lens. Dashed lines indicate approximate limits of physical tunability. ITO, indium tin oxide. To see this figure in color, go online.
D
B
PDMS hydrophilic liquid
Glass Slide ITO
SiO2 responsive hydrogel
Silicone oil hydrophobic coating
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Bates and Lu TABLE 1
Various Mechanisms for Employing Variable-Focus Lenses in Microfluidics and Their Advantages and Disadvantages
Tuning Mechanism Pneumatic
Advantages
Disadvantages
Application References
easy to integrate with existing microscopy systems
some arrangements require manual alignment of several device layers require high pressure source (~40 psi) long-term deformation and mechanical fatigue require external hardware and control electrode incorporation
adaptive focal length with large viewing angle (39)
large focal length tuning range can incorporate as in-plane (horizontal) or out-of-plane (vertical) configurations reasonable response time (100 ms) Electrowetting and dielectric forces
high lens-shape repeatability fast response times (50 ms)
Hyrodynamic
Responsive material
molecularly smooth interfaces many lens shapes possible
ability to alter lens shapes based on meaningful physiological change variety of lens shapes possible passive
open-air systems nonsterile, prone to evaporation require high voltages (~100 V), potential for hydrolysis only for planoconvex or planoconcave configurations significant external hardware and control required electrowetting: Joule heating and microbubbles slow response time (s) precise pressure control necessary (
Optics-Integrated Microfluidic Platforms for Biomolecular Analyses Kathleen E. Bates1,2 and Hang Lu1,2,* 1 Interdisciplinary Program in Bioengineering and 2School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia
ABSTRACT Compared with conventional optical methods, optics implemented on microfluidic chips provide small, and often much cheaper ways to interrogate biological systems from the level of single molecules up to small model organisms. The optical probing of single molecules has been used to investigate the mechanical properties of individual biological molecules; however, multiplexing of these measurements through microfluidics and nanofluidics confers many analytical advantages. Optics-integrated microfluidic systems can significantly simplify sample processing and allow a more user-friendly experience; alignments of on-chip optical components are predetermined during fabrication and many purely optical techniques are passively controlled. Furthermore, sample loss from complicated preparation and fluid transfer steps can be virtually eliminated, a particularly important attribute for biological molecules at very low concentrations. Excellent fluid handling and high surface area/volume ratios also contribute to faster detection times for low abundance molecules in small sample volumes. Although integration of optical systems with classical microfluidic analysis techniques has been limited, microfluidics offers a ready platform for interrogation of biophysical properties. By exploiting the ease with which fluids and particles can be precisely and dynamically controlled in microfluidic devices, optical sensors capable of unique imaging modes, single molecule manipulation, and detection of minute changes in concentration of an analyte are possible.
INTRODUCTION Despite the burgeoning activity in the field of optofluidics, there are many open opportunities to integrate optical tools with biological microfluidic handling systems to perform previously impossible analyses. Many optics-integrated microfluidic systems demonstrate comparable sensitivity to nonmicrofluidic systems, but most do not take full advantage of the potential to integrate sample handling and parallelize assays for ease of biological analysis. Assay systems that simplify and multiplex measurements and diagnostics could expand the reach and impact of optofluidic systems by making some types of high-throughput measurements possible and by making technology available to more diverse settings. Biological samples are diverse yet sparse solutions of cells, proteins, and small molecules, and are typically acquired in limited fluid volumes (milliliters to microliters), making them especially difficult to process. The issues of sample complexity and low molecular concentration, however, can be significantly decoupled from the problem of detection and quantification of analytes by incorporating microfluidic
methods. Myriad developments in microfluidics over the past two decades have demonstrated that biological samples can be successfully manipulated and separated from undesired components on-chip. The inherent heterogeneity in biological systems is a complicating factor that often requires the same assay be performed many times, a job that microfluidics are well suited for. In addition, many optical sensing systems can detect low concentrations of analyte, often with limits of detection in the pico- to attomolar range. In this review, we will attempt to provide the reader with an introduction to biological sample preparation in microfluidics as well as available cellular and subcellular techniques for optical manipulation and sensing in the hope to inspire new and interesting marriages between these fields. To this end, we will discuss manipulations and separations at molecular and cellular levels using optical techniques, hydrodynamics, and other force fields, in addition to optical tools commonly used in sensing systems, and optical sensing systems themselves. MATERIALS AND METHODS
Submitted October 13, 2015, and accepted for publication March 8, 2016. *Correspondence: [email protected] Editor: Brian Salzberg. http://dx.doi.org/10.1016/j.bpj.2016.03.018 Ó 2016 Biophysical Society
1684 Biophysical Journal 110, 1684–1697, April 26, 2016
Physical separations Separations are often required when working with biological samples to isolate a desired entity, sort a mixed population, or concentrate highly dilute
Optofluidics for Biomolecular Analyses mixtures. Because of the large variety of biological molecules that exist in cellular environments and the rarity of many of those molecules, separation is frequently the first step in biological analysis. By integrating these separation steps into a microfluidic system, it is possible to perform efficient and highly precise separations over a large range of particle sizes, from individual molecules and proteins (on the order of angstroms) to cells (on the order of micrometers) to small model organisms such as Caenorhabditis elegans (on the order of millimeters), in small volumes of liquid (on the order of nanoliters). Optical separations by themselves are limited by the analytical meaningfulness of optical differences. For example, the refractive index of individual cells within isogenic cell populations is often highly heterogeneous, depending on size and shape, making separation difficult. Consequently, although high-throughput and single-cell optical separation has proven very useful, there remain opportunities to improve the large-scale handling of particles. Before delving into more specialized optical separations, we will briefly mention some relevant nonoptical separations that supplement optical separations and could well be used to complement other optical sensing techniques. For a more thorough review of individual techniques, we point the reader to references cited in this section.
Nonoptical separations Within nonoptical separations, there are primarily two types of separation: those that occur passively due to hydrodynamic forces (pressure fields) within a microfluidic device, and those that occur due to imposed electrical or magnetic fields. As hydrodynamic forces are intrinsically required for microfluidic flow, they are particularly easy to integrate as a means of separation. The laminar nature of microfluidic flow results in a replicable system whose flow field can be modeled relatively simply, dramatically streamlining device design. Cells or other particles with different size, shape, or stiffness passing through a specifically designed flow field experience differential forces that direct them to different regions of the flow, allowing them to be separated passively. Applications are typically limited to populations where physical properties contrast significantly, but recent developments in inertial microfluidics have shown that micro- and nanoparticles can be separated in a high-throughput manner (600 ml/min) with high purity (90–99%), demonstrating potential for cell throughput close to the order of magnitude of flow cytometers (1,2). Although inertial microfluidics operate at higher Reynolds numbers, they remain laminar. Despite exposure of biologicals to higher shear stress compared with noninertial microfluidics, shear forces are much lower than what samples experience when they are pipetted, and separation in this manner may be gentler than centrifugation purification or physical filtration. Operation windows of inertial devices, however, are quite small and careful design is required (3). Microfluidic chromatography is another common method for which implementation is straightforward. High-resolution size separation over a large size range can be performed using deterministic lateral displacement, where regularly spaced features within the microfluidic channel bump different sized particles into different streamlines (4,5). For biological entities with a defined immunophenotype, microfluidic channel surfaces may be coated with antibodies before sample addition to perform affinity chromatography (6). The concept is simple, however, the requirement for a sample with a defined immunophenotype may be limiting, as cells must come into sufficient contact with channel walls to be captured, and antibodies are not only costly, but may also exhibit nonspecific binding. Externally applied fields may also be applied to microfluidic channels using principles of field flow fractionation (7). Separation by magnetic field is possible, but requires the use of magnetically responsive particles and antibodies in addition to an external magnetic field (6). Often the most useful technique in this category is dielectrophoresis (DEP) (8). Differences in dielectric properties of sample constituents allow continuous separation using an inhomogeneous electric field in DEP. The primary challenge of DEP is that many characteristics can impact the dielectric properties of the cell, including, but not limited to, cell size, and cell membrane and cytosolic content (which impacts permeability, capacitance, and conductivity). How-
ever, DEP has the significant advantage of being label-free and a reasonably sensitive method for separation despite its nonspecific nature (9–14). The applicability of DEP must be weighed carefully, as more complicated design considerations are involved, and exposure to an electrical field may damage or alter biological samples.
Optical property-based separations Label-free methods In general, optical manipulations operate on objects of approximately micron scale and have high spatial resolutions in addition to being labelfree and operating in a noncontact mode (15,16). Techniques that require labels have certain disadvantages; interference with molecular structures whose physical conformation is desired and interaction with biological processes of interest are both potential sources of error in labeled techniques, not to mention the added expense of additional reagents. The use of optical manipulation techniques on-chip often still requires bulky external equipment and patient optical alignment, however, external optics also allow optics to be decoupled from the microfluidics, enabling on-the-spot adjustment of key parameters rather than requiring further fabrication and redesign. Similar to DEP, optical manipulation can be very precise, but may require more complicated fabrication or alter cell physiology when highpowered lasers are involved. Investigating physical properties on the single-cell and single-protein level is an ideal application point for integrating optics and microfluidics. The use of optical tweezers (OT) to apply force, torque, and other fine-scale manipulations in a massively parallel fashion provides a window into the world of heterogeneity in both mechanical and optical senses. OTs use spatial light gradients to focus micron-sized particles to the center of a laser beam due to differences in radiation pressure. Classic examples are the measurement of the movement of the biological motor kinesin, and the characterization of the elasticity of DNA through attachment of these nanoscale objects to micrometer-sized spheres (17–19). Typical OT set-ups require high-power infrared lasers to focus particles, necessitating substantial modification to commercial microscopes and limiting the number of manipulable entities. The high-powered lasers needed could also lead to photodamage of biological molecules. Recently developed microfluidic alternatives for OTs have used plasmonic nanostructures to create evanescent waves at the interface of thin metal surfaces, generating a very strong near-field effect capable of trapping dielectric particles (20,21). Due to the relative system-level optical simplicity of optics on microfluidic systems in comparison to OTs, the localized nature of near-field effects, and the overall compactness of systems relying on surface plasmon resonance (SPR), plasmonics-based trapping lends itself to integration with microfluidic devices. SPR techniques on-chip is very promising for precise particle manipulations, including subdiffraction-limit trapping (22). Efficient assay parallelization leads to greater ease of sampling larger subsets of a population, thereby informing our perspective on heterogeneity. A current limitation for optical manipulations is the inefficiency of scaling up to many optical traps on a single microfluidic device. Although technologies like holographic optical tweezers and opto-electronic tweezers allow large-scale manipulation, these systems are expensive and optical contrast between different types of cells is often low, making manipulation difficult and again leading to the use of high power lasers (23,24). In a similar vein, projection of optical images onto microfluidic devices have been used to passively separate cells based on their optical contrast and polarizability (which increases as size increases), however, this relies on changes in optical contrast and polarizability being useful and meaningful ways to separate cells, which is not always the case (25). A technique that may enable high-throughput and precise manipulation on-chip has recently been described that employs lower power lasers than those typically required for multiplexing optical traps (26). In this case, multiple traps are created at antinodes of a standing wave from a single laser
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Bates and Lu and traps are repositioned by using thermal energy to change the phase of light waves. Rudimentary sorting has been performed by holding bead-conjugated biological samples in one set of traps while applying small lateral pressure fields to push untrapped particles out of the device, or alternatively, by flow switching based on Raman spectra obtained from single cells (16,26). This application relies heavily on the creation of highly stable traps that would experience significant drift outside of a well-defined microfluidic environment, and the ability to switch fluid flow quickly, a clear strength of microfluidic systems. Continued effort to create large arrays for simultaneous manipulation of individual particles in an efficient manner will likely remain a significant goal because of its potential impact on easing the investigation of heterogeneity and other fields reliant on big data for answers, for example genomic studies.
Labeled separations Images are one of the highest content data formats; a typical photosensor has several megapixels worth of data points. The conceptually simple idea of separation based on image properties and features is an integral part of many biological assays that can be supplemented through the use of microfluidic devices. By using microfluidic chips, micrometer-scale specimens can be imaged either in series or in parallel, both of which can enable high-throughput modalities (27). This is a common theme over a broad range of phenotyping applications, including both small organisms such as C. elegans as well as cells (28–30). The ability to restrict the movement of cells and model organisms while orienting asymmetrical objects reproducibly via hydrodynamic forces within microfluidic devices eases the time burden of physical manipulation as well as simplifies quantitative comparison of specimens. For living animals, various schemes are available for immobilization within devices, including cooling and compression, which are often much gentler compared with the usual glue or anesthetic methods (29,31). Because these chips are made of the flexible polymer polydimethylsiloxane (PDMS), chips are inexpensive to make, optically transparent, and compatible with biological specimens. The material properties of PDMS also enable on-chip membrane valves to conveniently control fluid flow. Although the use of microfluidics in this manner requires additional pressure sources and controls, time involved in operation and handling is orders of magnitude less compared with individually mounting and imaging animals or cells. Thus, for high-throughput experiments or experiments requiring large numbers of samples, as in genetic screens and phenotyping experiments, more comprehensive arrays of experiments come within reason for a single researcher to perform. Many separations based on image properties are facilitated by fluorescent labeling, but separation through image-based phenotyping is not exclusively confined to labeled reagents. Given that an object in a population to be sorted has sufficient regional variation that can be identified via brightfield, darkfield, or other modes of microscopy, these types of images can be used as the basis for separation. However, the use of transgenic cells and species that encode fusion proteins for reporter systems are common because of the ability to label proteins in subpopulations of cells in vivo. Subcellular localizations of proteins can often be determined as well (although how significantly fusion proteins affect localization is a matter of debate), and autofluorescence is typically low enough in most cells and organisms that differentiating background from foreground is relatively simple. Compared to a label-free system, a subcellularly localized reporter system can give much more detailed information about whole-system function because tagged proteins are actively involved in cellular processes. In addition, the number of reporters is theoretically only limited by the number of optically orthogonal reporters possible within the color spectrum, although in practice excitation and emission spectra often overlap significantly, making it impractical to use more than three or four reporters. This has recently driven the development of reporters that emit in nearinfrared range. Despite the usefulness of fluorescent reporters, the effort required to transform organisms and transfect cells can be significant, and damage due to photobleaching, as well as differences in the rate of photobleaching at different locations within the cell makes long-term moni-
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toring more challenging for parallelized methods (32). If intracellular or intraorganismal protein distribution is not required, this additional effort may not be warranted and label-free sensing may be more appropriate. On microfluidic chips, active separation of samples may involve acquisition of an image of the specimen, followed by actuating a valve on device based on optical feedback (i.e., based on specific image features), limiting the specimen to following a single path of least fluidic resistance. Recovery of specific specimens out of a large microfluidic array is sometimes only possible through the use of optical methods like OT that can provide control over individual specimens, however, this increases cost and power input significantly while requiring more user intervention (33). In contrast to most optical separation methods, using image features as criteria for separation is extremely versatile because separation need not be based upon a single feature, such as refractive index or physical size, but upon combinations of many features predefined by the user. Machine learning algorithms may be used to produce these combinations, limiting user-introduced bias toward phenotypes most evident to the human eye. However, even simple image processing followed by the application of heuristics relevant to the application of interest can be of use in situations where a desired phenotype is predefined (33). Although computer vision tools vary in terms of ease of use, there are many machine learning techniques that can be used out-ofthe-box or with little modification. The reader may consult Elicieri et al. (34) for more insight into image analysis packages and image processing and computer vision examples. In addition to consumer software available for computer vision, continuing innovations in computer vision algorithms have resulted in consistent improvement in terms of accuracy, speed, and bias reduction in the ability to detect image features in an unsupervised manner.
RESULTS Optical tools for guiding light on-chip In many optical sensing situations, the integration of multiple optical tools, such as lenses, waveguides, and lasers is critical to create a mechanism for sensing. Traditional optical systems for use with biomicrofluidics rely heavily on these components in the form of off-chip microscopy. In comparison, although most of the on-chip techniques described here are still dependent on external sensors (e.g., charge-coupled device or complementary metal oxide semiconductor sensors) to collect data, on-chip and partially on-chip optics confer advantages in terms of footprint, cost, and timescale while maintaining sensitivity expected from similar techniques. As many high-sensitivity optical techniques already require micro- or nanoscale structures to confine and propagate light, some type of microfabrication is already necessary in most cases. The addition of microfluidics, then, follows logically as a convenient way to control sample volume and handling without adding significantly more fabrication steps. On-chip lens systems Lenses are integral parts of most optical microscopy systems, but traditionally have little flexibility in terms of dynamic changes to focal length, necessitating precise translations in the z-direction to collect in-focus light from multiple image planes. In contrast to the rigidity of traditional lenses, cheaply produced on-chip lenses provide flexibility
Optofluidics for Biomolecular Analyses
both in terms of chip design and material properties, and can be used to facilitate on-chip imaging in a variety of ways. Practically, lenses are some of the easiest elements to integrate, as many common materials in microfabrication can be manipulated into cheap and robust lens arrays using simple techniques (35,36). Particularly in photometry applications, where wavefront fidelity is less important, simple converging and diverging lenses can be of use. Although variable-focus lenses were initially developed at the macroscale for use in spectacles, the fine scale at which they can be fabricated in microfluidics has yielded a small host of applications at the micron scale (37–39). Adjustable focal length microfluidic lenses are tunable by variation of refractive indices or lens shape. Refractive indices may be controlled through lens composition, or via thermal or electric fields, can be tuned pneumatically, through environmentally responsive materials such as hydrogels, or with electromagnetic fields (38–48). A primary challenge in fabricating variable focus lenses that function by lens deformation is in creating lenses with large dynamic ranges and short response times; for example, pneumatic control of fluid-filled reservoirs behind thin, flexible membranes (composed of, for example, PDMS) is a common microlens approach that is limited by slow response times despite large tuning ranges and easy device integration (Fig. 1 A) (49–51). Although PDMS is optically transparent and has low losses in the visible range, light scattering through the polymer is not always negligible due to nanoparticles of silica within commercially available products (52). Alternatively, nanoliter-size droplets can be easily, robustly, and precisely formed in microfluidic devices whose focal length can be tuned by altering the surface tension at the droplet interface or electrical field within the microfluidic device (43,49). Environmentally responsive lenses, such as hydrogels, have also been used to adjust focus in a passive
A
manner (see Fig. 1 D). In one interesting example, red blood cells were used as microlenses, switching their focal length from negative to positive values by changing the chemistry of the buffer solution and using their focusing properties as a basis for phenotyping (53). Using electrowetting to induce changes in lens geometry by altering the liquid contact angle has distinct advantages in consistency of fluid deformation, response time, and the need for only small driving voltages as compared with large pressures (Fig. 1 B) (45,46,54). The creation of arrays of these lenses on-chip is simplified by the ability to address individual droplets to spatial locations, either hydrodynamically or through the use of electrophoresis or electrowetting on dielectrics (EWOD). Temporal modulation of an electric field has recently been used to both create two-dimensional (2D) monodisperse droplet arrays for use as microlenses and to achieve large deformation of microlenses for variable-focus tunability over millisecond timescales, a speed similar to that of the inertial response limit that can be affected by a common piezoelectric z-stepper (49,55). In addition to these vertical tunable lenses, tunable lenses composed of core and cladding fluids within horizontal microfluidic channels also enable dynamic modulation of lens shape through hydrodynamics or electrokinetics (Fig. 1 C) (47,56–58). By exchanging fluids used for core and cladding, an additional parameter can be used for focal plane adjustment, further broadening the capabilities of this type of variable focus lens (59). Although these lenses are often easier to initially form, they can also be perturbed by small particles or bubbles within the flowing fluid, leading to significant chromatic and geometric aberrations and making them more difficult to maintain over long periods. Table 1 summarizes advantages and disadvantages of the primary types of dynamic lenses and example applications. Although acoustic tuning of microfluidic lenses has not been explored to our
cladding core
C
FIGURE 1 Physical configuration of variable focus lenses. (A) Pneumatically tuned lens. (B) Lens controlled by electrowetting. (C) Hydrodynamic lens. (D) Environmentally responsive lens. Dashed lines indicate approximate limits of physical tunability. ITO, indium tin oxide. To see this figure in color, go online.
D
B
PDMS hydrophilic liquid
Glass Slide ITO
SiO2 responsive hydrogel
Silicone oil hydrophobic coating
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Bates and Lu TABLE 1
Various Mechanisms for Employing Variable-Focus Lenses in Microfluidics and Their Advantages and Disadvantages
Tuning Mechanism Pneumatic
Advantages
Disadvantages
Application References
easy to integrate with existing microscopy systems
some arrangements require manual alignment of several device layers require high pressure source (~40 psi) long-term deformation and mechanical fatigue require external hardware and control electrode incorporation
adaptive focal length with large viewing angle (39)
large focal length tuning range can incorporate as in-plane (horizontal) or out-of-plane (vertical) configurations reasonable response time (100 ms) Electrowetting and dielectric forces
high lens-shape repeatability fast response times (50 ms)
Hyrodynamic
Responsive material
molecularly smooth interfaces many lens shapes possible
ability to alter lens shapes based on meaningful physiological change variety of lens shapes possible passive
open-air systems nonsterile, prone to evaporation require high voltages (~100 V), potential for hydrolysis only for planoconvex or planoconcave configurations significant external hardware and control required electrowetting: Joule heating and microbubbles slow response time (s) precise pressure control necessary (
